Method for separating and purifying Rubixanthin from pericarp of short-juvenile-phase wild citrus type arundina mandarin

Abstract

The invention belongs to the technical field of natural product extraction, and particularly relates to a method for separating and purifying Rubixanthin from pericarp of short-juvenile-phase wild citrus type arnica. The method at least comprises the following steps: S1, obtaining a total carotenoid crude extract from pericarp of Arnica mandarin; and S2, separating and purifying Rubixanthin from the total carotenoid crude extract by using semi-preparative UHPLC (Ultra High Performance Liquid Chromatography) with 100% methanol as a mobile phase A and 100% MTBE (Methyl Tert-Butyl Ether) as a mobile phase B. According to the invention, the Rubixanthin can be separated, identified and purified from the pericarp of the Arnica montana, and the substance with higher purity can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and particularly relates to a method for separating and purifying Rubixanthin from the peel of short-juvenile wild citrus fruit, Citrus aurantium. Background Art

[0002] Carotenoids are an important natural pigment widely found in nature. They are a class of tetraterpenes or triterpenes with isoprene as the basic skeleton. Due to the difference in the number of conjugated double bonds in the molecular structure, carotenoids present rich and colorful colors. In addition, carotenoids and their derivatives are also an important guarantee for human dietary nutrition and physical health. Among them, carotenoids containing β-ring structure (β-carotene and β-cryptoxanthin, etc.) are important precursors of vitamin A, and lutein in oxygen-containing carotenoids plays a very important role in human retinal health. In addition, other carotenoids have played an important role in improving human immunity, preventing cardiovascular diseases, preventing chronic diseases and cancer. As a natural pigment, carotenoids are important natural dyes in the fields of food cosmetics and textile production. Therefore, the large-scale industrial production of carotenoids has great application potential and economic value.

[0003] At present, the raw materials for industrial production of carotenoids mainly come from plant materials that are rich in carotenoids. The carotenoids that have been industrially extracted from plants mainly include lycopene, β-carotene, β-cryptoxanthin, lutein, and capsanthin, which have generated extremely high economic value in the fields of food and cosmetics.

[0004] As carotenoid research continues to deepen, people have gradually discovered that brightly colored carotenoids often have greater application potential, because they have more conjugated double bonds, are redder in color, and have stronger antioxidant capacity. For example, capsanthin extracted from peppers is stable and natural and harmless, and is widely used in food processing, cosmetic coloring, and drug sugar coating coloring agents. Capsanthin also has the effect of preventing radiation, especially the most significant effect of preventing γ-ray damage.

[0005] Other red carotenoids from plant sources include lycopene (mainly from tomatoes), β-limonene (mainly from citrus fruits), and rubixanthin (rose hips and sweet cherry). Rubixanthin was discovered in rose hips as early as 1934, and the exact structure of rubixanthin and its cis isomer gazaniaxanthin were determined in 1968 (Kuhn and Grundmann. On rubixanthin, a new xanthoyphyll of the formula C40H56O [J]. Berichte der Deutschen Chemischen Gesellschaft, 1934, 67: 339-344; Brown and Weedon. Rubixanthin and gazaniaxanthin [J]. Chemical Communications, 1968, 7: 382-384).

[0006] The Rutaceae citrus plants include the genera Citrus, Poncitrus and Fortunella. Common cultivated citrus (such as tangerines, grapefruits, oranges, mandarins, lemons, etc.) come from the genus Citrus. Fortunella hindsii is a single wild species in the genus Fortunella. It is an evergreen woody plant with an average fruit weight of 3-5g. The horizontal and vertical diameters of the fruit are both about 2cm, and the fruit surface is orange-yellow to dark red. Compared with common cultivated citrus, Fortunella hindsii has smaller fruits and shorter trees, so it is often called "mini citrus". The juvenile period of Fortunella hindsii is short, and seedlings can bloom in the same year and start to bear fruit in the second year. The juvenile period of common citrus seedlings is as long as 6-8 years. The research team of Huazhong Agricultural University has cracked the genome information of Fortunella hindsii and established a stable genetic transformation and gene editing system (Zhu C, Zheng X, Huang Y, et al. Genome sequencing and CRISPR / Cas9 gene editing of an early flowering Mini-Citrus (Fortunella hindsii) [J]. Plant biotechnology journal, 2019, 17 (11): 2199-2210.), which is expected to develop this variety into a model plant for citrus functional genomics research. Fortunella hindsii is edible in its entirety and is a potential nutritional and healthy functional citrus. In addition, the dwarfing characteristics of Fortunella hindsii make it suitable for facility cultivation in greenhouses, which is conducive to the development of supporting green production technologies to accurately control the content of active ingredients. It is a potential variety for realizing intelligent and unmanned production. Through large-scale screening of metabolites, the inventors found that the carotenoid composition of the mature peel of the kumquat is quite different from that of common citrus fruits. Only the peel of the kumquat accumulates a large amount of red Rubixanthin, which accounts for about 35.85% of the total carotenoids in the peel. It is the carotenoid with the highest content and the main reason for the red color of the kumquat peel.

[0007] In the prior art, only very small amounts of rubixanthin carotenoids and their analogs were detected in lemon peels and orange juices from the genus Citrus in the 1960s and 1970s, but they were not isolated and purified in large quantities (GROSS J, GABAI M, Lifshitz A. Carotenoids in juice of Shamouti orange [J]. Journal of Food Science, 1971, 36 (3): 466-473.; CURL AL. The carotenoids of Meyer lemons [J]. Journal of Food Science, 1962, 27 (2): 171-176.). Moreover, in recent decades, no studies have confirmed the presence of rubixanthin in lemons and oranges. Therefore, it is generally believed that citrus plants are not the source of rubixanthin. In the prior art, Rubixanthin is mainly isolated and identified from other plants (rose hips and sweet cherry), but there is no report on citrus plants (germplasm resources) that accumulate Rubixanthin in large quantities and the technology for extracting and purifying Rubixanthin in large quantities. In addition, compared with common β-carotene, β-cryptoxanthin has higher antioxidant activity and therefore has higher anti-inflammatory potential, which is very important for the human body to resist aging and prevent diseases (Katalin Sólyoma et al., Structure-response relationship of carotenoid bioaccessibility and antioxidant activity as affected by the hydroxylation and cyclization of their terminal end group [J], Food Research International, 2014, (66): 107-114.).

[0008] The inventors screened a large number of carotenoids from different wild citrus, closely related species of citrus, and cultivated citrus, and discovered for the first time that a short-lived wild citrus, Fortunella hindsii, can accumulate a large amount of red carotenoid Rubixanthin, giving it a bright red appearance, great ornamental value, and is an excellent material for large-scale purification of Rubixanthin. Other reported plant sources of Rubixanthin, such as cherries and rose hips, are not planted on a large scale, and citrus, as my country's largest fruit, will be enriched in Rubixanthin by improving its fruit in the future, which is expected to provide sufficient plant raw materials for the large-scale production of pure Rubixanthin, and will provide a material basis and technical guarantee for the innovative use of Rubixanthin natural pigments in the fields of health products and food cosmetics.

[0009] Rubixanthin, also known as 3R-β-carotene-3-ol, rubixanthin, has a molecular formula of C 40 H 56 O, CAS No: 3763-55-1, the structural formula is as follows:

[0010] Summary of the invention

[0011] In order to solve the deficiencies of the prior art, the present invention provides a method for separating and purifying Rubixanthin from the peel of a short-juvenile wild citrus - Kakultur (an independent species in the genus Fortunella). Through the present invention, Rubixanthin can be separated and purified in large quantities from the crude carotenoid extract of the Kakultur peel, and the obtained purity is relatively high.

[0012] The technical solution provided by the present invention is as follows:

[0013] A method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit, Citrus aurantium, comprises the following steps:

[0014] S1: crude extract of total carotenoids was obtained from the red peel of ripe fruits of C. fortunei;

[0015] S2: Rubixanthin was purified from the crude total carotenoid extract using semi-preparative UHPLC with 100% methanol as mobile phase A and 100% MTBE as mobile phase B.

[0016] The inventors have found that a large amount of rubixanthin can be accumulated in the pericarp of the fruit ...

[0017] Specifically, in step S1, the kumquat is a fully mature red kumquat fruit during the winter solstice in December, when the fruit changes from green to orange and then completely to scarlet. This time point is during the winter solstice in December (National Citrus Breeding Center, Wuhan, Hubei).

[0018] Specifically, step S1 includes the following steps:

[0019] S11: The red peel of the ripe fruit of the kumquat is quickly frozen with liquid nitrogen, and then freeze-dried and ground to obtain peel ground powder;

[0020] S12: Obtaining a crude extract of total carotenoids from the peel ground powder using a pigment extractant, wherein the pigment extractant is a mixed solution of n-hexane:acetone:anhydrous ethanol in a volume ratio of 2:(0.9-1.1, preferably 1):(0.9-1.1, preferably 1), and butylated hydroxytoluene (BHT) is added to the mixed solution at a mass fraction of 0.09-0.11‰.

[0021] Based on this technical solution, the pigment extractant is conducive to fully extracting carotenoids from the peel, and 2,6-di-tert-butyl-4-methylphenol (BHT) has an antioxidant effect, preventing carotenoids from being oxidized during the extraction process.

[0022] Specifically, in step S11, the peel ground powder is obtained by freeze-drying and then grinding, or by quick freezing with liquid nitrogen and then grinding.

[0023] Specifically, step S12 specifically includes the following steps:

[0024] S121: extracting the peel ground powder with the pigment extractant at 4-25° C. to obtain a pigment extract;

[0025] S122: washing the pigment extract with a saturated sodium chloride solution until it is neutral, discarding the aqueous phase, retaining the upper organic phase, and evaporating the solvent in the organic phase of the pigment extract to obtain a dry carotenoid crude extract;

[0026] S123: re-dissolving the crude carotenoid extract and performing a saponification reaction to convert the esterified carotenoid into a free form (non-esterified carotenoid);

[0027] S124: removing the aqueous phase from the saponification reaction product obtained in step S123, retaining the organic phase, washing the organic phase of the saponification reaction with a saturated sodium chloride solution until it is neutral, removing the aqueous phase, and evaporating the solvent to obtain the crude extract of total carotenoids in a free state.

[0028] Furthermore, step S1 also includes using HPLC to micro-separate and detect Rubixanthin from the carotenoid mixture; in the micro-separation and detection, 100% methanol is used as mobile phase A, methanol: water volume ratio = 4: (0.9 to 1.1, preferably 1) is used as mobile phase B, and 100% MTBE is used as mobile phase C.

[0029] Specifically, the micro-separation and detection of Rubixanthin was performed by the following HPLC method:

[0030] Chromatographic column: YMC C 30 Analytical chromatographic column (preferably 4.6 mm × 250 mm, 5 μm);

[0031] Mobile phase A: 100% methanol;

[0032] Mobile phase B: methanol: water = 4: (0.9-1.1, preferably 1);

[0033] Mobile phase C: 100% MTBE;

[0034] The flow rate is 1 ml / min;

[0035] Elution conditions: 0-6min 95% A; 6-7min 95-80% A; 7-12min 80% A for 5min; 12-32min 80-30% A; 32-48min 30% A for 16min; 48-50min 30-95% A; 95% A balance for 10min, always keep 5% B unchanged during elution; select the full wavelength range scan (200-600nm) for the visible light-ultraviolet absorption wavelength channel; the elution peak at 30-32min is Rubixanthin.

[0036] The peak eluting at 30-32 min of the chromatogram obtained by the HPLC-I method was identified as Rubixanthin, which was consistent with the standard. This method is recorded as HPLC-I.

[0037] Specifically, in step S2, a large amount of Rubixanthin is separated and purified by the following semi-preparative high performance liquid chromatography (UHPLC) method:

[0038] Chromatographic column: YMC C 30 Semi-preparative chromatography column (preferably 10 mm × 250 mm, 5.0 μm);

[0039] Mobile phase A: 100% methanol;

[0040] Mobile phase B: 100% MTBE;

[0041] Flow rate: 2 ml / min;

[0042] Elution conditions: 0-8 min 60% A; 8-28 min 60-40% A; 28-30 min 40% A for 2 min; 30-32 min 40-60% A; 32-37 min 60% A for 5 min;

[0043] Visible light / ultraviolet absorption channels: UV-vis-1: 450nm or UV-vis-2: 490nm;

[0044] The elution peak at 17.3-17.9 min was collected to obtain purified Rubixanthin.

[0045] The peak eluting at 17.3-17.9 min in the UHPLC chromatogram was identified as rubixanthin, and the purified rubixanthin was obtained by collecting the peak. This method was recorded as a UHPLC semi-preparative method.

[0046] Preferred: channels for visible light absorption are UV-vis-1: 450nm.

[0047] Furthermore, in step S2, the dried product of the crude free carotenoid extract is dissolved in 100% ethyl acetate, centrifuged at room temperature, and the supernatant is filtered through a 0.22 μm filter membrane, and then identified or purified.

[0048] Furthermore, the method further comprises step S3: performing purity analysis and mass spectrometry identification on the purified Rubixanthin;

[0049] In the purity analysis, 100% methanol was used as mobile phase A, methanol:water volume ratio = 4: (0.9 to 1.1, preferably 1) was used as mobile phase B, and 100% MTBE was used as mobile phase C;

[0050] In mass spectrometry identification, a mixture of methanol and water (99:1) containing 0.1% chromatographic grade formic acid was used as mobile phase A, and 100% MTBE was used as mobile phase B.

[0051] Specifically, the micro-separation and mass spectrometric identification of Rubixanthin were performed by the following LC-MS method:

[0052] The instrument was an ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS / MS) system (QExactive Plus, Thermo Fisher Scientific, San Jose, CA, USA) equipped with an atmospheric pressure chemical ionization (APCI) source and operated in positive and negative ion modes.

[0053] The parameters of the mass spectrometer are as follows: Full MS scan parameters: resolution of 70,000 half-peak width (FWHM); ion trap dynamic gain control (AGC) of 3e6; maximum injection time (IT) of 100ms; mass spectrum acquisition range of mass-to-nuclear ratio 100-1400m / z. ddMS2 scan parameters: 17,500FWHM; AGC of 1e5; minimum of 8e3; maximum IT of 50ms;

[0054] Chromatographic column: Thermo C 30 Chromatographic column (2.1mm×150mm,3.0μm):

[0055] Mobile phase A: methanol: water = 99:1 (containing 0.1% chromatography grade formic acid);

[0056] Mobile phase B: 100% MTBE;

[0057] Elution conditions (variable speed linear gradient elution): 0-6 min, 100% A, flow rate 0.25 ml / min; 6-6.5 min, flow rate increased to 0.5 ml / min; 6.5-15 min, linear gradient decreased to 80% A; 15-23 min, linear gradient decreased to 10%; 23-25 ​​min, maintained unchanged; 25-26 min, flow rate decreased to 0.25 L / min, linear gradient increased to 100% A.

[0058] The invention also provides a method for separating and purifying Rubixanthin in large quantities, and the Rubixanthin is extracted from the peel of the short-juvenile wild citrus fruit, the Citrus aurantium.

[0059] The inventors have found that the peel of the short-life wild citrus fruit, Citrus aurantium, can accumulate a large amount of rubixanthin, and Rubixanthin with a high purity can be separated and extracted in large quantities from Citrus aurantium.

[0060] The present invention also provides Rubixanthin obtained by the above methods, which has a purity greater than or equal to 97.62%.

[0061] Preferably, S1 comprises the following steps:

[0062] S11: Grinding the kumquat peel to obtain peel powder, preferably by vacuum freeze drying;

[0063] S12: Obtaining a crude extract of total carotenoids from the peel powder using a pigment extractant, wherein the pigment extractant is a 2:1:1 (v / v) mixed solution of n-hexane:acetone:anhydrous ethanol and contains 0.1‰ BHT by mass fraction.

[0064] Preferably, in S2, the HPLC method for detecting the presence or absence of Rubixanthin in the carotenoid sample of the fruit peel of the agaricus:

[0065] The chromatographic detection system was: Waters 2695 high performance liquid chromatograph, 2996 secondary array detector, 717 autosampler, Empower chromatography management software and YMC C 30 Analytical column (4.6 mm×250 mm, 5 μm) (Wilmington, NC, USA), 10 μl was injected each time.

[0066] Mobile phase A: 100% methanol;

[0067] Mobile phase B: methanol: water = 4:1;

[0068] Mobile phase C: 100% MTBE

[0069] The flow rate is 1 ml / min;

[0070] Elution conditions: 0-6min 95% A; 6-7min 95-80% A; 7-12min 80% A for 5min; 12-32min 80-30% A; 32-48min 30% A for 16min; 48-50min 30-95% A; 95% A for 10min, 5% B was kept constant during elution;

[0071] The elution peak at 30.5-31.5 min in the HPLC liquid chromatogram is Rubixanthin.

[0072] In this method, the elution peak at 30-32 min is Rubixanthin, which has a high content in the peel of the kumquat during the winter solstice ripening period and determines the formation of the red trait of the kumquat fruit.

[0073] Preferably, in S2, the HPLC method for separating, purifying, and extracting Rubixanthin in larger quantities is as follows:

[0074] Instrument: Dionex Ultimate 3000 semi-preparative HPLC equipped with Ultimate RS Variable Wavelength Detector.

[0075] Chromatographic column: YMC C 30 Semi-preparative chromatography column (preferably 10 mm × 250 mm, 5.0 μm);

[0076] Mobile phase A: 100% methanol;

[0077] Mobile phase B: 100% MTBE;

[0078] The flow rate is 2 ml / min;

[0079] Elution conditions: 0-8min 60% A; 8-28min 60-40% A; 28-30min 40% A for 2min; 30-32min 40-60% A; 32-37min 60% A for 5min. Select two visible light / ultraviolet absorption channels, UV-vis-1: 450nm, UV-vis-2: 490nm;

[0080] The elution peak at 17.3-17.9 min was collected to obtain purified Rubixanthin.

[0081] The method is suitable for further separating and purifying Rubixanthin from the total carotenoid extract of the fruit peel of the agaricus kumquat in larger quantities. The semi-preparative liquid phase used in the method has a higher flow rate, which greatly accelerates the speed of collecting the pure Rubixanthin product.

[0082] The beneficial effects of the present invention are as follows:

[0083] The invention identifies for the first time the Rubixanthin accumulated in large quantities from the fruit peel of the kumquat in the winter solstice mature period, separates and purifies it and extracts it in large quantities, and the obtained Rubixanthin has high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 The chromatogram is obtained by detecting the pigment components in the crude extract of total carotenoids using HPLC-I method.

[0085] Figure 2 Semi-preparative UHPLC chromatogram obtained during the separation process for the purification of Rubixanthin using the UHPLC semi-preparative HPLC method.

[0086] Figure 3 The chromatograms are obtained by HPLC detection, wherein Part A is a chromatogram obtained by HPLC detection of Rubixanthin separated and purified by the present invention, and Part B is a chromatogram obtained by HPLC detection of Rubixanthin standard. The two are basically consistent, indicating the accuracy of rubixanthin extracted by this method.

[0087] Figure 4 The first positive ion pattern of the mass spectrometry, wherein part A is the first positive ion pattern of the mass spectrometry of the Rubixanthin separated and purified by the present invention, and part B is the first positive ion pattern of the mass spectrometry of the Rubixanthin standard product. The two are basically consistent, indicating the accuracy of the rubixanthin extracted by the present method.

[0088] Figure 5 The mass spectrometry secondary pattern diagram is shown in Figure 1, wherein part A is the mass spectrometry secondary pattern diagram obtained by mass spectrometry analysis of the purified Rubixanthin standard, and part B is the mass spectrometry secondary pattern diagram obtained by mass spectrometry analysis of the Rubixanthin standard. The two are basically consistent, further demonstrating the accuracy of rubixanthin extracted by this method.

[0089] Figure 6 This is a comparison chart of different UHPLC separation effects. Part A in the figure: chromatogram of carotenoids separated from kumquat by UHPLC-I method. The black arrow indicates the position of Rubixanthin in the separation chromatogram. The small picture corresponds to the black arrow, which is the HPLC detection chromatogram after separation of the peak. Part B in the figure: chromatogram of carotenoids separated from kumquat by UHPLC-II method. The black arrow indicates the position of Rubixanthin in the separation chromatogram. The small picture corresponds to the black arrow, which is the HPLC detection chromatogram after separation of the peak. DETAILED DESCRIPTION

[0090] The principles and features of the present invention are described below. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0091] Unless otherwise specified, the test methods used in the embodiments of the present invention are all conventional methods; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0092] Fortunella hindsii Source: Huazhong Agricultural University National Citrus Breeding Center Resource Garden, December winter solstice, the color changes from green and orange to red or even scarlet. Fortunella hindsii can refer to the following literature: "Deng Xiuxin, Zhang Wencai. Study on direct induction of embryonic callus from Fortunella hindsii test tube seedlings. Fruit Trees in Southern China, 1988".

[0093] Example 1

[0094] 1. Extraction of carotenoids

[0095] 1) Grinding sample: Take a kumquat fruit (the kumquat fruit fully matures from green to orange and finally to scarlet on the winter solstice in December), cut the kumquat with a medical surgical scalpel, and scrape the pulp with a steel sample spoon to obtain the peel. After drying in a vacuum freeze dryer, grind the peel with a sample grinder (or mortar hand grinder) to obtain a dry peel sample, or directly freeze it with liquid nitrogen and grind it to obtain a wet peel sample.

[0096] 2) Pigment extraction: weigh 1 g of dry peel sample (3 g for wet sample) and place it in a 50 ml conical bottom centrifuge tube, add 35 ml of pigment extraction solution (n-hexane: acetone: anhydrous ethanol = 2:1:1, containing 0.1‰ BHT);

[0097] Add a 1.5 cm diamond magnetic rotor to each 50 ml conical bottom centrifuge tube containing the sample, shake it manually, and place it on a magnetic stirrer at 1500 rpm in a dark environment at 4 to 25 ° C for 2 hours;

[0098] Centrifuge at 4000 g for 15 min at room temperature, transfer the supernatant to a new 50 ml centrifuge tube, wash repeatedly three times with saturated NaCl (10%) aqueous solution until neutral, and discard the lower colorless aqueous layer;

[0099] The upper pigment solution was divided into 10 ml centrifuge tubes, and the vacuum concentrator was set to U-AQ mode, and the pigment extract was concentrated and evaporated at 30°C. After vacuum concentration, it was dissolved in 2 ml MTBE, and then 10% KOH-methanol solution (10 g KOH, 25 ml first-grade pure water, and 75 ml methanol) was added. After shaking and mixing, saponification was carried out at room temperature in the dark for 12 hours;

[0100] After adding 1 ml of MTEB to fully dissolve the pigment, add 2 ml of saturated NaCl aqueous solution to make it better separated, discard the lower water layer, and add 4 ml of saturated NaCl aqueous solution three times to wash until neutral. The pigment solution was divided into 10 ml centrifuge tubes, and the U-AQ mode of the vacuum concentrator was set at 30°C to vacuum concentrate and evaporate the pigment extract to dryness to obtain a crude carotenoid extract.

[0101] 2.HPLC detection

[0102] All the extracted carotenoid crude extracts were dissolved in ethyl acetate (chromatographic grade), centrifuged at 4°C, 12000 rpm for 30 min, and then filtered through a 0.22 μm microporous filter membrane. The filtered liquid was transferred to the inner insert tube in the sample loading bottle and prepared for detection. In this example, HPLC-I method was used for detection.

[0103] The chromatographic detection system was: Waters 2695 high performance liquid chromatograph, 2996 secondary array detector, 717 autosampler, Empower chromatography management software and YMC C 30 Analytical column (4.6mm×250mm, 5μm), 10ul injection each time.

[0104] The mobile phase was: phase A: 100% methanol; phase B: methanol: water = 4:1; phase C: 100% MTBE. Elution conditions: 0-6min 95% A; 6-7min 95-80% A; 7-12min 80% A for 5min; 12-32min 80-30% A; 32-48min 30% A for 16min; 48-50min 30-95% A; 95% A for 10min, and 5% B was kept constant during elution.

[0105] The results are as follows Figure 1 As shown in the HPLC liquid chromatogram, the elution peak at 30.5-31.5 min indicated by the arrow is Rubixanthin, and the small figure on the right shows its visible light / ultraviolet absorption peak.

[0106] 2. Purification of Rubixanthin

[0107] Large-scale separation and purification of carotenoid Rubixanthin: The above carotenoid dry material was dissolved in 100% ethyl acetate (chromatographic grade) organic solution, centrifuged at room temperature, 12000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter membrane and added to the sample loading bottle. The instrument model and chromatographic conditions used for semi-preparative separation of Rubixanthin are as follows. In this example, semi-preparative UHPLC method was used for separation and purification.

[0108] Instrument: Thermo Scientific Dionex Ultimate 3000 semi-preparative HPLC, equipped with Ultimate RS Variable Wavelength Detector. Column: YMC C 30 Semi-preparative column (10 mm × 250 mm, 5.0 μm);

[0109] Mobile phase A: 100% methanol;

[0110] Mobile phase B: 100% MTBE;

[0111] The flow rate is 2 ml / min;

[0112] Elution conditions (multi-step gradient elution): 0-8min 60% A; 8-28min 60-40% A; 28-30min 40% A for 2min; 30-32min 40-60% A; 32-37min 60% A for 5min. Select two visible light / ultraviolet absorption channels, UV-vis-1: 450nm, UV-vis-2: 490nm;

[0113] The eluted fractions were collected according to their peak order and peak time, dried in a vacuum concentrator at 30 degrees, and the vacuum concentrator was set to U-AQ mode to obtain purified Rubixanthin. Figure 2 As shown, the absorbance value at 450nm is shown. It can be seen from the figure that the peak time of Rubixanthin is about 16.9-19.2min (indicated by the arrow). After calculation, about 91.74μg of pure Rubixanthin can be extracted and purified from every 1g of dried peel sample.

[0114] 3. Purity analysis and mass spectrometry identification

[0115] 1) Purity analysis

[0116] A small amount of the prepared dried product and Rubixanthin standard were dissolved in an appropriate amount of ethyl acetate, centrifuged at room temperature and 12000 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter membrane before sampling and detection. This method uses a Waters 2695 high performance liquid chromatograph from Waters Company for purity detection.

[0117] The chromatographic detection system was: Waters 2695 high performance liquid chromatograph, 2996 secondary array detector, 717 autosampler, Empower chromatography management software and YMC C 30 Analytical column (4.6mm×250mm, 5μm), 10ul injection each time.

[0118] In this example, HPLC-I method was used for detection

[0119] Mobile phase A: 100% methanol;

[0120] Mobile phase B: methanol: water = 4:1;

[0121] Mobile phase C: 100% MTBE

[0122] The flow rate is 1 ml / min;

[0123] Elution conditions: 0-6min 95% A; 6-7min 95-80% A; 7-12min 80% A for 5min; 12-32min 80-30% A; 32-48min 30% A for 16min; 48-50min 30-95% A; 95% A for 10min, 5% B was kept constant during elution;

[0124] The results are as follows Figure 3 As shown in FIG. 1 , the purity of the Rubixanthin obtained in the present invention is very high, and a single main peak is shown in the HPLC detection, and the purity of the Rubixanthin obtained in the present invention is very high ... 40 H 56 O, Cas number: 3763-55-1), and the color of pure Rubixanthin is orange-red. The calculated purity is 97.62%.

[0125] 2) Mass spectrometry identification

[0126] The purified Rubixanthin and the standard were subjected to mass spectrometry analysis, respectively. The instrument used was an ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS / MS) system (Q Exactive Plus, Thermo Fisher Scientific, San Jose, CA, USA) equipped with an atmospheric pressure chemical ionization (APCI) source and operated in positive and negative ion modes.

[0127] The parameters of mass spectrometry are as follows: Full MS scan parameters: resolution 70,000 half-peak width (FWHM); ion trap dynamic gain control (AGC) 3e6; maximum injection time (IT) 100ms; mass spectrum acquisition range 100-1400 m / z. ddMS2 scan parameters: 17,500 FWHM; AGC 1e5; minimum 8e3; maximum IT 50ms.

[0128] Using Thermo C 30 Chromatographic column (2.1mm×150mm, 3.0μm), 1ul injection each time.

[0129] The mass spectrometry identification LC-MS method used in this example is as follows:

[0130] Mobile phase A: methanol: water = 99:1 (containing 0.1% chromatography grade formic acid);

[0131] Mobile phase B: 100% MTBE;

[0132] Elution conditions (variable speed linear gradient elution): 0-6 min, 100% A, flow rate 0.25 ml / min; 6-6.5 min, flow rate increased to 0.5 ml / min; 6.5-15 min, linear gradient decreased to 80% A; 15-23 min, linear gradient decreased to 10%; 23-25 ​​min, maintained unchanged; 25-26 min, flow rate decreased to 0.25 L / min, linear gradient increased to 100% A.

[0133] The data were analyzed using the accompanying Xcalibur 3.0 software (Thermo Fisher Scientific, San Jose, CA, USA).

[0134] The results are as follows Figure 4 and Figure 5 As shown, the molecular weight of Rubixanthin obtained in the present invention is 553.426, and the secondary mass spectrum information is also consistent with the standard (C 40 H 56 O, Cas number: 3763-55-1), indicating that the substance isolated from the golden orange is Rubixanthin.

[0135] Example 2

[0136] Rubixanthin was purified by referring to the above Example 1, except that the dry powder of the peel of the short-juvenile wild citrus fruit of the species Citrus acuminata (a separate wild species different from other citrus) was used as the raw material for extraction. As a result, about 91.74 μg of pure Rubixanthin with a purity of not less than 97% was obtained per gram of dry weight of Citrus acuminata peel.

[0137] Comparative Example 1

[0138] Rubixanthin was purified with reference to the above Example 1, except that the carotenoids in the Jaffa orange juice were separated into hydrocarbons, monohydric alcohols, dihydric alcohols and polyhydric alcohols by column chromatography (MgO-Hyflo Super Cel, filled with a mass ratio of 1:1, eluted with a mixture of ether and acetone), and then the carotenoids in different fractions were separated and identified by thin layer chromatography (calcium chloride-silica gel G, silica gel G as adsorbent, light petroleum, benzene, ether, acetone, ethyl acetate, dichloromethane, polyethylene and isopropanol and a large amount of organic reagents were used for analysis). After the cumbersome and large amount of highly polluting organic reagent separation and identification, only 0.05% of the total carotenoid content of Rubixanthin was detected, and the purity was unknown (GROSS J, GABAI M, Lifshitz A. Carotenoids in juice of Shamouti orange [J]. Journal of Food Science, 1971, 36 (3): 466-473.). Previous researchers separated and identified carotenoids from 500g, 1100g, and 1800g of Meyer lemons. They used n-hexane-99% methanol (v:v=1.8:1) in a Craig instrument and performed up to 200 separations to fractionate different carotenoids. They only identified Rubixanthin-like, which accounted for 0.8% of the total carotenoid content, but did not provide mass spectrometry or nuclear magnetic resonance analysis information, so it was impossible to determine whether it was Rubixanthin (CURL AL. The carotenoids of Meyer lemons [J]. Journal of Food Science, 1962, 27 (2): 171-176.). In short, the content of Rubixanthin / Rubixanthin-like detected by previous researchers in Jaffa orange juice and Meyer lemon peel was very low, and it was basically impossible to separate and purify in large quantities and industrially extract them. The present method uses a chromatographic column as an adsorption carrier, methanol and MTBE as eluents, greatly reducing the amount of organic reagents used, and for the first time identified Rubixanthin in the mountain kumquat, and the Rubixanthin content in the red peel of the mature fruit of the mountain kumquat is as high as 35.85%, about 700 times that of the orange juice of Jaffa orange, and more importantly, the purity of Rubixanthin separated and prepared by the present method is as high as 97.62%. In addition, the raw materials mentioned in previous studies, Jaffa orange and Meyer lemon, are all hybrid offspring between different species in the genus Citrus. In contrast, the mountain kumquat used in the present invention is a unique wild species in the genus Fortunella. Therefore, the present invention realizes the identification, separation and purification of Rubixanthin in the genus Fortunella for the first time.

[0139] Comparative Example 2

[0140] UHPLC-I Method:

[0141] (1) Mobile phase:

[0142] Phase A: methanol: MTBE = 1:1;

[0143] Phase B: methanol: water: MTBE = 6: 3: 1;

[0144] Flow rate: 2ml / min;

[0145] (2) Elution conditions (gradient elution)

[0146] 0-20min 92%A; 20-23min 92-82%A; 23-25min 82%-50%A; 25-26min 50%-40%A; 26-36min 40%A maintained for 10min.

[0147] Visible light / ultraviolet absorption channel selection: UV-vis-1: 450nm;

[0148] (3) Separation results

[0149] The elution peaks at 21.5-22.1 min in the UHPLC-I chromatogram were collected and identified by HPLC. In addition to the chromatographic peak of Rubixanthin, there was another chromatographic peak at the same height, and Rubixanthin could not be separated well.

[0150] UHPLC-II method (same as semi-preparative UHPLC used for purification above):

[0151] (1) Mobile phase

[0152] Phase A: 100% methanol;

[0153] Phase B: 100% MTBE;

[0154] Flow rate: 2ml / min;

[0155] (2) Elution conditions (gradient elution)

[0156] 0-8min 60%A; 8-28min 60-40%A; 28-30min 40%A for 2min; 30-32min

[0157] 40-60% A; 32-37 min 60% A for 5 min.

[0158] Visible light / ultraviolet absorption channel selection: UV-vis-1: 450nm;

[0159] (3) Separation results

[0160] The peak eluting at 17.3-17.9 min in the UHPLC-II chromatogram was collected and identified as Rubixanthin with high purity.

[0161] Comparison of UHPLC-I and UHPLC-II methods:

[0162] like Figure 6 As shown, the UHPLC-II method removes the aqueous phase and changes it to a pure organic phase, which enhances the separation of carotenoids, and the elution conditions are further simplified to a gradient elution of 60-40% A. Since the mobile phase in the UHPLC-II method is changed to a pure organic phase, the collected elution peak solvent does not contain water, making it easier to evaporate and dry to obtain pure Rubixanthin.

[0163] The above are only preferred embodiments of the present invention and are not intended to limit the present invention in any form. Any ordinary technician in the industry can smoothly implement the present invention as shown in the drawings and above. However, any equivalent changes, modifications and evolutions made by technicians familiar with the profession without departing from the scope of the technical solution of the present invention using the technical content disclosed above are all equivalent embodiments of the present invention. At the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the protection scope of the technical solution of the present invention.

Claims

1. A method for separating and purifying Rubixanthin from the peel of the short-lived wild citrus fruit, Citrus aurantium, characterized in that: The following steps are involved: S1: crude extract of total carotenoids was obtained from the peel of the fruit of the fruit of the genus Acuminata; S2: Rubixanthin was purified from the crude total carotenoid extract using semi-preparative UHPLC with 100% methanol as mobile phase A and 100% MTBE as mobile phase B.

2. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of the species Acuminata according to claim 1, characterized in that: In step S1, the kumquat is a red kumquat fruit that is fully ripe during the winter solstice in December; Step S1 includes the following steps: S11: The peel of the kumquat is quickly frozen with liquid nitrogen, and then freeze-dried and ground to obtain peel ground powder; S12: Obtaining a crude extract of total carotenoids from the peel ground powder using a pigment extractant, wherein the pigment extractant is a mixed solution of n-hexane:acetone:anhydrous ethanol in a volume ratio of 2:(0.9-1.1):(0.9-1.1), to which butylated hydroxytoluene is added at a mass fraction of 0.09-0.11‰.

3. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of the species Acuminata according to claim 2, characterized in that: In step S11, the peel ground powder is obtained by freeze-drying and then grinding, or by quick freezing with liquid nitrogen and then grinding; Step S12 specifically includes the following steps: S121: extracting the peel ground powder with the pigment extractant at 4-25° C. to obtain a pigment extract; S122: washing the pigment extract with a saturated sodium chloride solution until it becomes neutral, discarding the aqueous phase, retaining the upper organic phase, and evaporating the solvent in the organic phase of the pigment extract to obtain a crude carotenoid extract; S123: re-dissolving the crude carotenoid extract and performing a saponification reaction to convert the esterified carotenoid into a free form; S124: removing the aqueous phase from the saponification reaction product obtained in step S123, retaining the organic phase, washing the organic phase of the saponification reaction with a saturated sodium chloride solution until it is neutral, removing the aqueous phase, and evaporating the solvent to obtain the crude carotenoid extract in a free state.

4. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Achyranthes fortunei according to claim 1, characterized in that: Step S1 also includes using HPLC to micro-separate and detect Rubixanthin from the carotenoid mixture; in the micro-separation and detection, 100% methanol is used as mobile phase A, methanol: water volume ratio = 4: (0.9-1.1) is used as mobile phase B, and 100% MTBE is used as mobile phase C.

5. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Achyranthes fortunei according to claim 4, characterized in that: In step S1: The micro separation and detection of Rubixanthin was carried out by the following HPLC method: Chromatographic column: YMC C 30 Analytical columns; Mobile phase A: 100% methanol; Mobile phase B: methanol: water = 4: (0.9-1.1); Mobile phase C: 100% MTBE; The flow rate is 1 ml / min; Elution conditions: 0-6min 95% A; 6-7min 95-80% A; 7-12min 80% A for 5min; 12-32min 80-30% A; 32-48min 30% A for 16min; 48-50min 30-95% A; 95% A for 10min, 5% B was kept constant during elution; The elution peak at 30-32 min is Rubixanthin.

6. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Kaempferia fortunei according to claim 1, characterized in that: In step S2, a large amount of Rubixanthin is separated and purified by semi-preparative high performance liquid chromatography (UHPLC) as follows: Chromatographic column: YMC C 30 Semi-preparative columns; Mobile phase A: 100% methanol; Mobile phase B: 100% MTBE; Flow rate: 2 ml / min; Elution conditions: 0-8min 60% A; 8-28min 60-40% A; 28-30min 40% A for 2min; 30-32min 40-60% A; 32-37min 60% A for 5min; Visible light / ultraviolet absorption channels: UV-vis-1: 450nm or UV-vis-2: 490nm; Collect the elution peak at 17.3-17.9 min to obtain purified Rubixanthin; In step S2, the dried product of the crude carotenoid extract is dissolved in 100% ethyl acetate, centrifuged at room temperature, and the supernatant is filtered through a 0.22 μm filter membrane, and then separated and purified.

7. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Achyranthes fortunei according to claim 1, characterized in that: The step S3 includes: performing purity analysis and mass spectrometry identification on the Rubixanthin obtained by separation and purification; In the purity analysis, 100% methanol was used as mobile phase A, methanol:water volume ratio = 4:(0.9-1.1) was used as mobile phase B, and 100% MTBE was used as mobile phase C; In mass spectrometry identification, a mixture of methanol and water (99:1) containing 0.1% chromatographic grade formic acid was used as mobile phase A, and 100% MTBE was used as mobile phase B.

8. The method for separating and purifying Rubixanthin from the peel of the short-juvenile wild citrus fruit of Kaempferia fortunei according to claim 7, characterized in that: The micro separation and mass spectrometric identification of Rubixanthin were performed by the following LC-MS method: The instrument is: an ultra-high performance liquid chromatography-high resolution tandem mass spectrometry system equipped with an atmospheric pressure chemical ionization ion source, and operating in positive and negative ion modes; The parameters of mass spectrometry are as follows: Full MS scan parameters: resolution of 70,000 half-peak width; ion trap dynamic gain control of 3e6; maximum injection time of 100ms; mass spectrum acquisition range of mass-to-nuclear ratio 100-1400m / z; ddMS2 scan parameters: 17,500FWHM; AGC of 1e5; minimum of 8e3; maximum IT of 50ms; Chromatographic column: Thermo C 30 Chromatographic column: Mobile phase A: a mixture of methanol containing 0.1% chromatographic grade formic acid and water = 99:1; Mobile phase B: 100% MTBE; Elution conditions: 0-6 min, 100% A, flow rate 0.25 ml / min; 6-6.5 min, flow rate increased to 0.5 ml / min; 6.5-15 min, linear gradient reduced to 80% A; 15-23 min, linear gradient reduced to 10%; 23-25 ​​min, maintain unchanged; 25-26 min, flow rate reduced to 0.25 L / min, linear gradient increased to 100% A.

9. A method for extracting Rubixanthin, characterized in that: Extracted from the red peel of the ripe fruit of the short-lived wild citrus species, Citrus aurantium.

10. An extract obtained according to the method according to any one of claims 1 to 9, characterized in that: The purity of Rubixanthin is greater than or equal to 97.62%.

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